High resolution high performance liquid chromatography fingerprinting of purified human chorionic gonadotropin demonstrates that oxidation is a cause of hormone heterogeneity.
To characterize the structures of the various forms of hCG and its immunologically related fragments measured in blood, urine, and tissue culture media, we developed a highly sensitive HPLC tryptic fingerprinting system that can characterize as little as 15 micrograms of material. Standard preparations of the subunits of hCG purified from pregnancy urine were found to exhibit heterogeneity on reverse phase HPLC chromatography, complicating efforts to characterize such forms of hCG. The same type of chromatographic heterogeneity was observed when the hormone was reduced and S-carboxymethylated (RCM) and its RCM subunits separated on HPLC. One major and one minor peak were observed in the alpha- and beta-subunits from both native and RCM hormone, with a ratio of between 5:1 and 10:1. Development and application of a high resolution, semimicrobore HPLC fingerprint technique used together with an examination of selected peptides by amino acid sequencing and mass spectrometry defined the differences between these forms as oxidation. Using each of the isolated RCM subunit's major and minor HPLC peaks, the oxidation was shown to be reversible and probably due to interconversion of methionine with its sulfoxide form.
Pollak
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Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032.
Halpine
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Chait
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Birken
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D006063:Chorionic Gonadotropin / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D010449:Peptide Mapping / Q000379:methods*; D014357:Trypsin
D006063:Chorionic Gonadotropin; D014357:Trypsin